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REVIEW published: 11 February 2016 doi: 10.3389/fevo.2016.00008

The Incredible Lightness of Being Methane-Fuelled: Stable Isotopes Reveal Alternative Energy Pathways in Aquatic Ecosystems and Beyond

Jonathan Grey *

Lancaster Environment Centre, Lancaster University, Lancaster, UK

We have known about the processes of methanogenesis and methanotrophy for over 100 years, since the days of Winogradsky, yet their contributions to the carbon cycle were deemed to be of negligible importance for the majority of that period. It is only Edited by: in the last two decades that methane has been appreciated for its role in the global Jason Newton, carbon cycle, and stable isotopes have come to the forefront as tools for identifying Scottish Universities Environmental Research Centre, UK and tracking the fate of methane-derived carbon (MDC) within food webs, especially Reviewed by: within aquatic ecosystems. While it is not surprising that chemosynthetic processes David Bastviken, dominate and contribute almost 100% to the biomass of organisms residing within Linköping University, Sweden Blake Matthews, extreme habitats like deep ocean hydrothermal vents and seeps, way below the reach Eawag: Swiss Federal Institute of of photosynthetically active radiation, it is perhaps counterintuitive to find reliance upon Aquatic Science and Technology, MDC in shallow, well-lit, well-oxygenated streams. Yet, apparently, MDC contributes to Switzerland varying degrees across the spectrum from point sources to extremely diffuse sources. *Correspondence: Jonathan Grey Certainly a good proportion of the evidence for MDC contributing to freshwater food [email protected] webs comes from somewhere in the middle of that spectrum; from studies of seasonally stratifying lakes (mono- or dimictic) wherein, there is a defined gradient or boundary Specialty section: This article was submitted to at which anoxic meet oxic conditions and consequently allows for close coupling of Population Dynamics, methanogenesis and methanotrophy. However, even seemingly well-mixed (polymictic) a section of the journal Frontiers in Ecology and Evolution lakes have a contribution of MDC contributing to the benthic biomass, despite an almost continual supply of photosynthetic carbon being delivered from the surface. Aside Received: 31 October 2015 Accepted: 25 January 2016 from the fundamental importance of identifying the carbon sources fuelling biomass Published: 11 February 2016 production, stable isotopes have been integral in the tool box of palaeolimnologists Citation: seeking to identify how contributions from methane have waxed and waned over time. Grey J (2016) The Incredible Lightness of Being Methane-Fuelled: Stable Here, we synthesize the current state of knowledge in the use of stable isotopes to trace Isotopes Reveal Alternative Energy MDC in primarily freshwater ecosystems. Pathways in Aquatic Ecosystems and Beyond. Front. Ecol. Evol. 4:8. Keywords: methanotrophy, chironomids, zooplankton, carbon, food webs, greenhouse gas, biogeochemical doi: 10.3389/fevo.2016.00008 cycling, trophic transfer

Frontiers in Ecology and Evolution | www.frontiersin.org 1 February 2016 | Volume 4 | Article 8 Grey Identifying and Tracking Methane in Food Webs

A BRIEF SYNOPSIS ON THE GLOBAL routes by which CH4 produced in anoxic freshwater sediments IMPORTANCE OF METHANE IN AQUATIC may either by-pass or become incorporated into food webs is SYSTEMS, AND PARTICULARLY IN shown in Figure 1. FRESHWATERS COULD A METHANE PATHWAY BE The global carbon cycle was considered, until relatively recently, IMPORTANT TO SECONDARY to be solely the flux and storage of carbon between the PRODUCTION IN FOOD WEBS? atmosphere, and terrestrial and oceanic pools. Within the total carbon budget, it has been noted that despite their relatively Anoxic water and sediments are typically rich in organic small area, inland freshwaters make a considerable contribution matter compared to the overlying oxic water, and anoxic to the global methane (CH4) budget with emissions of CH4 from metabolism may account for a substantial part (20–60%) of the freshwaters being at least comparable to the terrestrial CH4 sink carbon metabolism and the heterotrophic microbial production (Battin et al., 2009). However, there is a considerable bias toward within freshwater environments (Hessen and Nygaard, 1992). data from lakes and other wetlands, and the role of rivers remains Methanogenesis in lakes has been reported corresponding to 30– poorly defined (Bastviken et al., 2011). Emissions of CH4 may be 80% of the anaerobic mineralization in waters and sediments small in terms of carbon, but one must consider that CH4 is a (Bastviken, 2009). While seasonal variability in CH4 oxidation is more potent greenhouse gas than CO2 over century time scales; known to be considerable, especially in dimictic lakes, between (Bastviken et al., 2011) estimated that global CH4 emissions 30–94% of the CH4 reaching oxygenated layers is reputedly expressed as CO2 equivalents correspond to at least 25% of the oxidized (Casper et al., 2000; Morana et al., 2015). In essence estimated terrestrial greenhouse gas sink. Our understanding of then, CH4 is a major product of the C mineralization in the global carbon cycle will only be complete if we include the lakes, and a large proportion may be converted to microbial flux of carbon through inland freshwaters (Cole et al., 2007; biomass equivalent in some instances to the total C fixation by Battin et al., 2009; Trimmer et al., 2012) getting to grips with heterotrophic bacteria and a significant proportion of primary methane-fuelling of food webs is an interesting and important production (Hessen and Nygaard, 1992; Bastviken et al., 2003). component of this. Indeed, (Cole, 2013) noted that “the role of Again, data from rivers are lacking, but across 15 rivers, in late methane in supporting food webs in lakes, and perhaps even summer, i.e., when one might expect the greatest contribution beyond their shores, has come as a surprise” and that “the from photosynthesis, (Shelley et al., 2014) conservatively notion that lake methane partially supports higher organisms in calculated that net methanotrophy was equivalent to between 1 surrounding terrestrial environments fundamentally changes our and 46% of benthic net photosynthetic production within the understanding of how aquatic food webs work.” gravel beds of chalkstreams. Couple this to the apparently high Methanogenesis is a universal terminal degradation process (50%) carbon conversion efficiency of methanotrophs (relative of organic matter in anoxic aquatic sediments when inorganic to 10–30%, typical for bacteria in detrital-based food webs), oxidants such as nitrate, ferric iron, or sulfate are depleted regardless of marked spatial and temporal changes in ambient (Conrad, 2005). Hence, in marine systems where there is methane concentration, and it suggests that methanotrophs can typically a high concentration of sulfate, the sulfur cycle sustain net production throughout the year (Trimmer et al., tends to dominate chemosynthesis, but in freshwaters where 2015). sulfate concentrations are typically lower (Hobbie, 1988) then The importance of a CH4 pathway to food webs might yet methanogenesis dominates. Stable isotopes have been an increase further under climate change. Increases in temperature incredibly useful tool in the identification and quantification forecast for the coming decades may have profound implications of methanogenic and methanotrophic pathways (Conrad, 2005) for the cycling of carbon in aquatic ecosystems due to the and further identifying the constituents of the complex microbial differential temperature dependencies of carbon fixation by community that is actively involved via stable isotope probing gross primary production (GPP) and carbon mineralization (SIP; e.g., He et al., 2012), but those aspects are not the focus of by ecosystem respiration (ER). For example, (Yvon-Durocher this review. Methane may be lost directly from the system via et al., 2010) showed that warming of 4◦C reduced the carbon ebullition or the recently hypothesized micro-bubble pathway, sequestration capacity of freshwater mesocosms by 13%, shifting stochastic processes notoriously difficult to quantify (Prairie and them toward net heterotrophy (i.e., net sources of CO2 to del Giorgio, 2013) or be effectively “piped” to the surface via the atmosphere) because ER responded more strongly to plants (Bergstrom et al., 2007; Sanders et al., 2007). Alternatively, temperature than GPP. They also found that methanogenesis or in addition, it may subsequently serve as an energy and C responded even more strongly than ER or GPP, with 20% more ◦ source for methanotrophs (methane oxidizing bacteria; MOB), of the GPP being accounted for by CH4 emissions with 4 C typically at oxic-anoxic boundaries (if anaerobic CH4 oxidation of warming (Yvon-Durocher et al., 2011). Benthic community is excluded) in the sediment, or in the water column (Kankaala structure and how that contributes to a host of ecosystem et al., 2007). It is essentially from this point in the cycle that processes, including microbial and macrofaunal decomposition stable isotopes have been key in tracing the use of methane- rates, was also clearly affected by such warming (Dossena et al., derived carbon (MDC) into, and through, food webs, particularly 2012). If it is assumed that delivery of organic matter does not in freshwaters (Jones and Grey, 2011). A schematic of potential change but temperature increases as predicted, then for example,

Frontiers in Ecology and Evolution | www.frontiersin.org 2 February 2016 | Volume 4 | Article 8 Grey Identifying and Tracking Methane in Food Webs

FIGURE 1 | Methane produced in anoxic sediments may be routed through plants (1) or lost from the sediments to the atmosphere via ebullition or micro-bubbles (2). If it reaches a boundary with oxygen at the sediment-water interface (under mixed or weakly stratified conditions), MOB oxidize it and create biomass which routes via benthic macroinvertebrates into benthic, pelagic, and terrestrial predators (3). Under strongly or permanently stratified conditions, methane will diffuse upwards through the water-column, and oxygen (and MOB) might first be encountered at the metalimnion, where zooplankton link MDC into higher predators (4). An indirect route for MDC could be via CO2 derived from the oxidation of CH4 might then be cycled through phytoplankton, and hence on to zooplankton (5), or indeed via sedimentation back down to benthic macroinvertebrates.

the increased mineralization will equate to a 4–27% (0.9–6.4 Tg (δ13C value from C3 plants typically −28 to −26‰; Peterson Cy−1) decrease in organic carbon burial in boreal lakes (Gudasz and Fry, 1987) or autochthonous phytoplankton [δ13C typically et al., 2010). However, very recent work in rivers suggests between −35 to −25‰;(Grey et al., 2000; Vuorio et al., 2006) but that methanotrophy has the potential to match methanogenesis acknowledging that components of the phytoplankton such as enhanced by warming (Shelley et al., 2015). How climate change their fatty acids may be ∼10‰ further 13C-depleted (e.g., Taipale 13 might impact upon food web mediation of MDC will be returned et al., 2015)]. However, CH4 δ C values reported from sediments to later. are not necessarily linked to the δ13C values of sedimentary organic matter; instead they may be strongly influenced by the quality of the organic matter substrate and/or the predominant WHY ARE STABLE CARBON AND methanogenic pathway (Rinta et al., 2015), and of course to HYDROGEN SUCH USEFUL TRACERS OF a certain extent as to whether some of the CH4 has already METHANE? been oxidized by MOB prior to analysis (Coleman et al., 1981). In marine hydrocarbon seep communities, δ13C has been the 13 12 Isotopic signatures of environmental CH4, both C/ C and primary isotope value examined, used to differentiate between 2H/1H, have been compiled by Whiticar et al. (1986) and Bréas animals with chemoautotrophic symbionts (–40 to –20‰) from et al. (2001) amongst others. An important characteristic of those with methanotrophic symbionts (≤–40‰; Brooks et al., biogenic methane is that its carbon stable isotope composition 1987) and to identify the source CH4 pool as either thermogenic 13 13 13 is typically markedly C-deplete compared to other putative (δ C = −45 to −40‰) or biogenic (δ C < −45‰) CH4 13 basal resources in a food web. So, for freshwater lakes, CH4 δ C (Sassen et al., 1999). ‰ may be as low as −110 to −50 dependent upon formation Isotopic fractionation during the use of CH4 by MOB typically pathway; (Whiticar, 1999; Deines and Grey, 2006; Taipale et al., leads to further 13C-depletion (by 0–30‰; Summons et al., 2007) relative to either allochthonous terrestrial plant detritus 1994; Templeton et al., 2006). For example, CH4-consuming

Frontiers in Ecology and Evolution | www.frontiersin.org 3 February 2016 | Volume 4 | Article 8 Grey Identifying and Tracking Methane in Food Webs archaea isolated from anoxic marine sediments have been reported with δ13C values as low as −96‰ (Orphan et al., 2001), while biomarkers (e.g., archaeol and hydroxyarchaeol) from such archaea within a CH4-supported benthic microbial community in cold-seep sediments exhibited δ13C values as low as −111‰ (Werne et al., 2002). Hence, the MOB biomass available to consumers has a strikingly low δ13C and, because stable carbon isotope ratios differ little between consumers and their diets, assuming no selective assimilation or substantial biosynthesis (McCutchan et al., 2003; Grey, 2006), this should allow its contribution to consumer biomass to be rather readily traced. Hydrogen isotope effects during methanogenesis of methylated substrates can lead to deuterium depletions as large as −531‰, whereas, bacterial D/H discrimination for the CO2-reduction pathway is significantly less (−250 to −170‰; Whiticar, 1999). Very little is known regarding the δD values of MOB. However, when compared to typical values of autochthonous (−290 to −215‰) and allochthonous (−160 to −125‰) resources, there is still great scope for the use of δD to trace CH4-derived production (Estep and Dabrowski, 1980; Doucett et al., 2007), especially when in combination with δ13C (e.g., Deines et al., 2009). The more distinct the sources, and indeed, the more tracers used, the more confidence can be assigned to estimates of contribution to diet derived from any of the recently published mixing models (e.g., Parnell et al., 2013). Problems arise using isotopic tracers when a relatively minor contribution from MDC results in δ values that could be arrived at via alternative pathways (see Section “The Zone of Contention” below).

METHANE USE ACROSS A SPECTRUM OF SOURCES

As appreciation of the possibility of MDC providing an alternative energy source to food webs has grown, so the emphasis on research has shifted from point sources to ever more diffuse sources, and less intuitively obvious locations where it might be relevant. The proportion of MDC contributing to food webs at more diffuse sources may well be smaller (but still of FIGURE 2 | Stable isotope bi-plots of food webs across a spectrum of significance); as such, there is likely to be greater ambiguity in point to diffuse sources of methane with corresponding decrease in the stable isotope signal, and so the importance of MDC might strength of δ13C value as a tracer of methane-derived carbon (MDC); have been overlooked in many of these systems (Figure 2). blue boxes indicate components of the food web with small / It is perhaps unsurprising that the use of CH4 (amongst negligible influence of MDC. (A) Gulf of Alaska (redrawn with permission other chemosynthetic production) is strongly evident at point from Levin and Michener, 2002): solid symbols—pogonophoran field infauna; open symbols—clam field infauna. (B) Plußsee (strongly stratifying small lake, sources such as deep-sea vents and seeps, whale, kelp and wood data from Harrod and Grey, 2006): open circles—fish; solid falls, and some sewage outflows, typically far beyond the direct circles—macroinvertebrates; open squares—chironomid larvae. (C) Loch reach of photosynthetically active radiation (although of course Ness (weakly stratifying large lake, data from Jones and Grey, 2011): open photosynthetic production can “fall-out” of the water column to circles—fish; solid circles—invertebrate and basal resources; open benthic communities). The potential for chemosynthesis to fuel squares—chironomid larvae. entire animal communities in the ocean was first noted around 35 years ago (e.g., Rau and Hedges, 1979). Early attention focused on megafaunal or epifaunal taxa such as molluscs or pogonophorans but there was a broad suite of smaller infaunal deposit feeding the likes of Van Dover and Fry (1994), Colaço et al. (2002), and and omnivorous invertebrates whose mode of nutrition remained Levin and Michener (2002). For example, Levin and Michener largely ignored until the application of stable isotope analyses by (2002) looked at a variety of sites including CH4 seeps in the Gulf

Frontiers in Ecology and Evolution | www.frontiersin.org 4 February 2016 | Volume 4 | Article 8 Grey Identifying and Tracking Methane in Food Webs of Alaska, on the Oregon margin, and on the northern California inter-individual variability that has been observed in chironomid slope and found that seep macrofauna exhibited lighter δ13C stable isotope ratios (e.g., Grey et al., 2004a; Figure 2). (and δ15N) values than those in non-seep sediments. Significant When stratification of the water column becomes too contributions were found from MDC to macrofaunal biomass pronounced, generally in duration, and the benthic sediments from sediments of pogonophoran fields (32–51%) and clam beds become inhospitable even for the hypoxic tolerant chironomid (12–40%) in the Gulf of Alaska, and in microbial mat sediments larvae, Jones and Grey (2011) hypothesized that MDC is on the Oregon margin (20–44%). Some polychaetes exhibited more likely to be taken up in the water column at the extremely low values of δ13C (−90.6‰) at these point sources oxic-anoxic boundary by zooplankton. Again, evidence for (see Figure 2A). this is mostly derived from the field from small Finnish Within the last 15 years, research on MDC and food webs boreal lakes with marked oxyclines (e.g., Jones et al., 1999; primarily focussed on lakes, particularly stratifying lakes in Taipale et al., 2007, 2008; but see Bastviken et al., 2003; temperate and boreal systems, and much of this work has Santer et al., 2006; Schilder et al., 2015a). Pelagic zooplankton been extensively reviewed by Jones and Grey (2011). Tube- δ13C values are typically not as low as those reported dwelling chironomid larvae appear key in lake sediments. Field from similar lakes for benthic chironomids, perhaps again studies from lakes across Alaska, England, Finland, and Germany reflecting the more diffuse nature of the source CH4, and/or (amongst others) have demonstrated that chironomids can the more mobile feeding capability of zooplankton in the assimilate MDC extensively (up to 70% of larval biomass; Jones water column relative to tube dwelling chironomids in the et al., 2008). The degree to which they do may vary within lakes sediments. Some of the lowest values reported are for on a temporal (Grey et al., 2004b; Deines et al., 2007b) or spatial Daphnia spp. from small, strongly stratifying lakes with anoxic (Deines and Grey, 2006; Gentzel et al., 2012) scale, or by taxa hypolimnia; for example, −47‰ in a kettle lake, Plußsee (Jones and Grey, 2004; Kelly et al., 2004; Jones et al., 2008), and (Harrod and Grey, 2006), or −46‰ from Mekkojarvi (Taipale among lakes with “strength” of stratification (Grey et al., 2004b; et al., 2008). Laboratory support for zooplankton uptake Deines et al., 2007a; Hershey et al., 2015). Chironomid larvae of MOB is sparse, but (Kankaala et al., 2006) measured are bioengineers; they bioturbate the sediment while “digging” growth rates of Daphnia in replicated cultures fed microbial and maintaining their burrows and draw down oxygenated suspensions with or without addition of CH4 and found water, bringing it into contact with anoxic sediment. The that their δ13C values indicated consumption of 13C-depleted sediments on the burrow walls have been shown to exhibit MOB, as have (Deines and Fink, 2011) using 13C-labeling higher methane oxidation rates and higher densities of MOB than of CH4. the surrounding bulk or surficial sediments (Kajan and Frenzel, Evidence of MDC contributions to biomass in polymictic 1999; Gentzel et al., 2012). Larvae thereby appear to create the (permanently mixed) lakes is rarer. Such lakes are often perfect micro-niche for the coupling of methanogenesis and shallow and contain considerable stands of macrophytes; while methanotrophy (Deines et al., 2007a; Kelly et al., 2004; see route methanogenesis is certainly proceeding in the sediments, much 3 in Figure 1). of the CH4 produced might be routed via the plant stems It was assumed from field studies that the low δ13C values for and via ebullition (routes 1&2 in Figure 1) and hence, side- taxa such as Chironomus plumosus (e.g., −70 to −50‰; Jones step incorporation into the food web [although see reference et al., 2008) reflected ingestion of the MOB on their burrow walls to Agasild et al. (2014), below]. Since the whole water column (Deines et al., 2007a) akin to “gardening” by trichopteran caddis is well oxygenated, there is no distinct boundary where MOB flies on the biofilms that develop on caddis cases (also studied by will accumulate and thus it is unlikely that zooplankton will 13 using stable isotopes; (Ings et al., 2012). By using C-labeled CH4 feed heavily upon MOB (Jones and Grey, 2011). In the benthos, additions directly into sediments housing chironomid larvae there is also typically a more consistent supply of 13C-enriched under controlled experimental settings, Deines et al. (2007a) phytoplankton production from above which will “swamp” the have elegantly demonstrated that larvae assimilate MDC via lower δ13C values from MOB. Examples of such lakes with MOB; this was further supported by phospholipid fatty acids permanently oxic sediment surface layers in which MDC has diagnostic for MOB and significantly enriched by the 13C-labeled been shown to make only a limited (maximum ∼20%) or methane being detected in the larval tissues. In a series of parallel negligible contribution to chironomid biomass include Großer experiments, they showed that larvae could also obtain MDC Binnensee and Schöhsee in north Germany (Grey et al., 2004b; via 13C-labeled Type II MOB introduced into the water column Deines et al., 2007b), Lough Neagh and Rostherne Mere in the above sediments. Type I and Type II MOB use different pathways UK (Kelly et al., 2004), Izunuma in Japan (Yasuno et al., 2012), for formaldehyde assimilation (ribulose monophosphate and and Võrtsjärv in Estonia (Agasild et al., 2014; Cremona et al., serine, respectively), and typically favor different environmental 2014). Interestingly, the latter lake was sampled at various sites conditions; Type I appear to be dominant in environments and it was only at one particular site dominated by vegetation 13 in which CH4 is limiting and combined nitrogen and copper that low δ C values were recorded in both zooplankton and concentrations are relatively high, whereas Type II appear where chironomids. Agasild et al. (2014) postulated that the stands there are high CH4 concentrations, low dissolved oxygen, and of macrophytes prevented wind mixing from disturbing the limiting concentrations of combined nitrogen and/or copper sediments, and that dissolved oxygen in the water column was (Hanson and Hanson, 1996). The ability to access MDC via reduced by the restricted circulation of water and gas exchange two discrete routes might account for some of the incredible between the water surface and the atmosphere and by increased

Frontiers in Ecology and Evolution | www.frontiersin.org 5 February 2016 | Volume 4 | Article 8 Grey Identifying and Tracking Methane in Food Webs oxygen demand from the decomposition of organic matter; all by emerging chironomid imagos from stratifying lakes (Jones processes which would lead to greater MDC being available to and Grey, 2011). The potential is clear to see for vertebrate the food web. predators as well, such as barn swallows (Hirundo rustica) which, Within the last 5 years has come the first convincing using stable isotopes, have been identified as prioritizing such evidence of MDC contributing to food webs in free-flowing, well abundant prey at specific times of the year (Parnell et al., 2013). oxygenated streams and rivers, where because of the turbulent Of course, we should also consider how alteration of a food web, nature, the source of CH4 could be considered to be most diffuse. for example by introduction of a top predator for recreation or One of the first studies claiming a river food web to be fuelled by as a function of range expansion might cause cascading effects MDC was by Kohzu et al. (2004) who reported Helodes sp. beetle down to biogeochemical cycling near the base of a food web. By larvae and adults with δ13C values as low as −69.8‰ but these experimentally manipulating fish density in a previously fish-less were from stagnant backwater pools akin to stratifying lakes, and lake, (Devlin et al., 2015) showed that a trophic cascade from while these may be important habitats on some lotic systems, fish to microbes affected methane efflux to the atmosphere and they were not from the free flowing, main-stem river food web. reduced the amount of MDC assimilated into the biomass of Since then, considerable research on the chalk streams of the UK, zooplankton that remained (assessed from Daphnia δ13C values). highly productive, ground water fed systems has revealed that It may well be that such improved quantitative understanding trichopteran larvae may play a similar role to chironomids in of the influence of higher trophic consumers on carbon budgets lakes, the main conduit for MDC to route into the wider food creates future opportunity for management and policy to identify web (e.g., Trimmer et al., 2009, 2010). In contrast, Mbaka et al. and implement new options for mitigating greenhouse gas release (2014) studied small inline impoundments with extremely short at regional scales (Schmitz et al., 2014). residence times on a river system in Germany but could find negligible evidence of MDC contributing to chironomids from THE ZONE OF CONTENTION the sediments there. How MDC might contribute significantly to river food webs clearly requires more research. Various authors (e.g., Deines et al., 2009) have acknowledged Unless there is almost 100% trophic transfer of MDC higher that confidence in the use of isotopic tracers of MDC from field into the food web, then obviously mixing with non-MDC food studies must be tempered where/when alternative explanations sources results in a dilution of the indicator isotope in question, for such isotope values can arise. The “zone of contention” for and the ability to trace MDC further using stable isotopes alone δ13C from consumers in freshwater lakes for example typically is weakened (see below). An apparent gradient is thus evident occurs between −40 and −30‰. Chironomid larvae could from point to diffuse source of methane. For example, on a exhibit such a value if they assimilated: (a) a small percentage species-specific basis, some mobile benthic predators (eels, sea from very low δ13C MOB and a greater percentage from relatively stars, and predatory snails) have been shown on the basis of high δ13C phytoplankton (e.g., Grey et al., 2004a); (b) alternative their low δ13C (and δ15N& δ34S) values to obtain close to 100% chemosynthetic sources of carbon such as sulfur bacteria (e.g., of their nutrition from CH4 seep production in the Gulf of Deines et al., 2009; Roach et al., 2011); or (c) phytoplankton with Mexico (MacAvoy et al., 2002). From stratifying lakes, (Harrod very low δ13C. It should be remembered that these scenarios are and Grey, 2006) and (Ravinet et al., 2010) have found isotopic not mutually exclusive. Scenario c may arise because a substantial evidence of MDC contributing (up to ∼12%) to bream (Abramis part of the dissolved CO2 pool may originate from respiration of brama) and to ruffe (Gymnocephalus cernuus), respectively, autochthonous and allochthonous organic matter and have low while in a shallow, well-mixed Pantanal (tropical) wetland lake δ13C (from −20 to −15‰; Lennon et al., 2006; Kankaala et al., (Sanseverino et al., 2012) could trace MDC into various fish 2010). The degree of fractionation of that CO2 by phytoplankton species. Even from the very shallow lake Võrtsjärv, Agasild et al. is uncertain and extremely variable, but in lakes might range (2014) reported that at sites amongst the macrophytes where from 0 to 15‰ (with values near the upper end of the range zooplankton and chironomid larvae were most 13C-deplete, probably most widespread; (Bade et al., 2006). Therefore, it is there was a corresponding decrease in δ13C for roach (Rutilus not uncommon to find δ13C values for lake phytoplankton of rutilus), perch (Perca fluviatilis), and the apex predator, pike < −30‰ (e.g., Grey et al., 2000; Vuorio et al., 2006), and (Esox lucius), indicative of trophic transfer of MDC to the very anything feeding selectively on 13C-depleted phytoplankton (or top of the food web. To date, evidence from rivers has not been assimilating selectively from components thereof such as fatty reported, but given the extremely abundant nature of the primary acids) will show correspondingly low δ13C values (Pel et al., consumers (particularly cased caddis flies) that appear key to 2003). The same has been shown for rivers (Finlay et al., 1999). linking MOB into the food web in such systems, the pathway The situation is even more complex when a proportion of the low 13 is certainly in place (Trimmer et al., 2012). Evidence of the δ C values for CO2 could have originated from the oxidation transfer of MDC across ecosystem boundaries is still limited. of CH4, and hence in effect, be an indirect contribution from Aquatic invertebrates such as Helodes sp., Chloroperlidae spp., MDC (Route 5 in Figure 1). Further dilution of the MDC Leuctridae spp., and Sialis sp. have all been recorded from Malaise signal with trophic transfer up the food web has already been traps on stream banks, i.e., post emergence, with δ13C values mentioned. from −69.8 to −51.8‰ (Kohzu et al., 2004) but there has still In such scenarios, only with the addition of alternative but been only one study quantifying transfer of MDC and that was complementary tracers can the assimilation of MDC be assigned into riparian spiders (up to 18% of their biomass) mediated with confidence. Hence, the addition of further stable isotopes

Frontiers in Ecology and Evolution | www.frontiersin.org 6 February 2016 | Volume 4 | Article 8 Grey Identifying and Tracking Methane in Food Webs such as δD [e.g., (Deines et al., 2009; Belle et al., 2015; van preserved in most lake sediment records. Chironomid remains, Hardenbroek et al., 2016), δ34S(Grey and Deines, 2005), and to especially the larval head capsules, can be found abundantly a certain extent δ15N (Stephen et al., 2002; Grey et al., 2004a); in lake sediments. Indeed, exoskeleton fragments originating see later discussion], have proved useful in ascertaining the use from molting and deceased larvae, or zooplankton resting eggs, of MDC. Radio isotopes might offer some support under certain are preserved for tens to hundreds of thousands of years at a situations; for example. (Opsahl and Chanton, 2006) studied quality which allows microscopic identification usually to genus, the food webs of troglobitic organisms in the Upper Floridian or species morphotype, but sometimes also to species level (van aquifer and found that crayfish trapped from remote sinkhole Hardenbroek et al., 2011). Since lake sediments can be dated conduits were not only on average ∼10‰ 13C-depleted relative using radiometric and/or other dating methods, these remains to their counterparts at accessible springs at the surface but that can be used to reconstruct historical community composition there was a strong correlation with radiocarbon (14C) depletion and by inference the water quality, ambient temperature, or a relative to modern values, indicative of a chemosynthetic food particular habitat structure (Eggermont and Heiri, 2012). Head source. Concurrent analysis of phospholipid fatty acids (PLFAs) capsules and exoskeletons comprise mainly chitin and proteins which are diagnostic for MOB, as well as compound-specific and, on the basis that their chemical composition does not seem analysis of the isotope ratios of those PLFAs has also been to be strongly affected by decomposition processes, they can invaluable. For example, (Taipale et al., 2009) demonstrated a be used to develop palaeo-environmental reconstructions based strong relationship between the δ13C values of Daphnia and the upon stable isotope composition (Perga, 2010, 2011; Heiri et al., proportion of MOB-specific PLFAs in Daphnia. These methods 2012). have also highlighted the indirect route via methane-oxidation Heiri et al. (2012) recently reviewed the available stable isotope 13 and uptake of the resulting C-depleted CO2 by autotrophs studies based on fossil chironomids (which had mainly examined (Route 5 in Figure 1). For bog-pool food webs in Estonia, the elements C, N, H, and O), and identified four key areas: (1) (Duinen et al., 2013) suggested that the most parsimonious developing the methodology for preparing samples for isotopic explanation for relatively low δ13C values of algae-derived analysis, (2) studies rearing chironomid larvae under controlled polyunsaturated fatty acids found in insects was that MOB were laboratory conditions to determine those factors affecting stable creating CO2 from the oxidation of CH4 which was assimilated isotope composition, (3) ecosystem-scale studies relating stable by their direct algal “neighbors” within the biofilm community. isotope measurements of fossil chironomid assemblages to (Sanseverino et al., 2012) used the presence of MOB-diagnostic environmental conditions, and (4) developing the first down- PLFAs in various benthivorous and detritivorous fishes to core records describing past changes in the stable isotope support claims of MDC assimilation in Brazilian wetlands as composition of chironomid assemblages. In a relatively short the fish δ13C values were <−36‰; low relative to the other period of time since that review, a number of publications have food web components in question but clearly not the marked arisen expanding upon those areas, further demonstrating the 13 C-depletion classically associated with CH4. Further correlative usefulness of the technique, and including other complementary evidence may be provided by concurrent assessment of the tracer evidence to support suppositions when the stable isotopes methanogen/methanotroph community by molecular methods. alone might prove ambiguous. Eller et al. (2005) reported zones of aerobic and anaerobic CH4 Firstly, it is important to determine whether there is any oxidation in the water column of a strongly stratifying lake, where isotopic offset between the recalcitrant parts of organisms high cell numbers of MOB were also detected by fluorescence recovered from palaeolimnological samples and the whole in situ hybridization techniques. It was around this depth in body that is typically analyzed for the study of contemporary the same lake that (Santer et al., 2006) found that one of the relationships in food webs. It is also important to determine cyclopoid copepod species, Diacyclops bicuspidatus, consistently whether the “clean up” protocols that palaeo-samples typically maintained highest density and exhibited δ13C values ∼10‰ require have any significant effect upon isotopic integrity. To lower than epilimnetic species and the photosynthetic particulate answer both of these questions for chironomid head capsules, organic matter sampled during the same time interval, and (van Hardenbroek et al., 2010) trialed various commonly used proposed the role of MDC in its diet. chemical methods for sediment processing and found that treatment with 10% KOH, 10% HCl, or 40% HF showed no LOOKING BACK: HINDCASTING detectable effect on δ13C, whereas, perhaps unsurprisingly, a combination of boiling, accelerated solvent extraction and heavy A particular area of research related to CH4-fuelling of food chemical oxidation resulted in a small but statistically significant webs that has emerged most recently aims to identify or decrease in δ13C values (0.2‰). Then, using a modification of determine past “methane environments,” predominantly in the culturing experimental protocol by Deines et al. (2007a), 13 lakes. Insight into past variations of CH4 availability in lakes they demonstrated with MOB grown on C-labeled methane, would further our understanding of the timing and magnitude that methanogenic carbon is transferred into chironomid head of the response of lake CH4 production and emissions to capsules (van Hardenbroek et al., 2010). Frossard et al. (2013) changing environmental conditions. Palaeolimnologists have have also looked at head capsule to whole organism isotopic long recognized that recalcitrant remains of organisms such as offsets for chironomid larvae and reported from experimental the strongly sclerotized head capsules of chironomids or the rearing on three different diets that the head capsules were 13C- ephippia of daphniids, can be found in abundance and well depleted by 0.9‰ relative to whole biomass. For zooplankton,

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Perga (2010) has shown that the C and N stable isotope after AD 1970, which coincided with periods of warmer climate compositions of the daphniid exoskeleton and those of the (indicated by an independent local temperature record). As a 13 13 whole body are strongly correlated. Exoskeleton δ C values were consequence, the discrepancy in δ C between CH4-sensitive similar to those of the whole body but were strongly depleted in taxa and bulk organic matter was higher in these sections 15N(−7.9‰), reflecting its derivation from excretory ammonia than in other parts of the core, whereas the δ13C of other of dietary origin, known to be 15N-depleted compared with invertebrate taxa did not show the same trend. They concluded dietary organic nitrogen (Schimmelmann, 2011). Further elegant that there was higher CH4 availability in the study lake during experiments have shown that the stable isotopic composition of warmer periods and that the energy sources of some key benthic Daphnia ephippia provides information on that of the parent invertebrates changed accordingly. Wooller et al. (2012) managed Daphnia, and of the food and water they were exposed to to reconstruct the CH4 history of Qalluuraq Lake, a shallow during formation. Schilder et al. (2015b) demonstrated that Alaskan tundra lake, over a period ∼12,000 years in this manner, there were only small offsets between Daphnia and ephippia and similar work has been conducted on large, deep sub-alpine relative to the range of variation in Daphnia stable isotopic lakes, particularly in France. A change from oligotrophic status composition reported from down-core studies. Interestingly associated with anthropogenic nutrient enrichment over the however, their work also indicated that temperature may have last 150 years was examined for associated shifts in the basal a minor influence on the δ13C, δ15N, and δ18O values of resources available to the benthic food web (Frossard et al., 2015). Daphnia body tissue and ephippia which has implications for Chironomid head capsule δ13C values started to decrease with the water temperature reconstruction work using oxygen isotopes, onset of eutrophication in both Lake Annecy and Lake Bourget; as well as highlighting the care with which controlled feeding the estimates of the MDC contribution to chironomid biomass experiments need to be conducted (sensu Perga and Grey, ranged from <5% prior to the 1930s to nearly 30% in recent 2010). The suite of organism remains has been further extended years. recently, as it now appears bryozoan statoblasts and zooids have To date, values for chironomid head capsules have not been the potential to act as indicators of MDC (van Hardenbroek et al., reported as 13C-depleted as for live organisms. This is in part 2016). a frustrating function of the requirement for multiple head Prior to the interest in palaeo-reconstruction, site-specific, capsules to be pooled to provide sufficient material for elemental and hence, differing CH4 production potential and oxidation and isotopic analysis. It is also likely associated with the fact that had only been linked to living chironomid larvae (e.g., Deines the sampling of the remains of organisms at a specific location and Grey, 2006). More confidence in the potential of recalcitrant (depth) might not truly reflect the location where the animal remains to provide information about past changes in CH4 assimilated its diet, due perhaps to resuspension of sediments availability in lakes using sediment records has arisen since and/or focussing of material (Battarbee, 1999). Hence, the studies have been conducted across lake types and actually “strength” of a MDC signal that one can find in a contemporary using remains from surficial sediments i.e., reflecting the most sample derived from fresh larvae with values for individuals 13 recent CH4 history that can be measured concurrently. In a <−70‰, will always be dampened (i.e., less C-depleted) by study of seven Swedish lakes, (van Hardenbroek et al., 2012) pooling and/or dilution effects in palaeolimnological samples. observed significant negative correlations between the δ13C As a consequence, the usefulness of δ13C alone as a tracer of Chironomini and both CH4 fluxes at the lake surface, deteriorates (see Section The Zone of Contention above). One and CH4 releases from the sediment. That dataset was built very promising approach is the analysis of ancient DNA (aDNA) upon by incorporating samples from 10 Siberian lakes and from the methanotroph community. Belle et al. (2014) has expanding the suite of remains to include those of Daphnia elegantly demonstrated how aDNA can be used to complement and Tanytarsini; the δ13C of all three groups were correlated stable isotopes in a study of a sediment core from the deepest significantly with diffusive CH4 flux in the combined Siberian zone of Lake Narlay, representing the last 1500 years of sediment and Swedish dataset suggesting that δ13C in the biomass of these accumulation. A significant change was noted since ca AD1600, invertebrates was affected by CH4 availability (van Hardenbroek with an increase in the proportion of MOB in the total bacteria et al., 2013). Schilder et al. (2015a) measured Daphnia ephippial community, and a corresponding decrease in chironomid head δ13C values from the surface sediments of 15 small European capsule δ13C. These trends suggest that assimilation of MOB lakes, and found a strong correlation to the late summer aqueous may account for up to 36% of chironomid biomass, with CH4 concentration in both the surface water and above the evidence for preferential assimilation of methanotroph type I sediment. and the NC10 phylum. Parallel strands of evidence are clearly Down-core work is providing some tantalizing evidence of required whenever there is ambiguity in stable isotope data, and past CH4 variability over time. Adding to their proof-of-concept the development of aDNA will surely grow in this particular work on which invertebrate remains are useful tracers of MDC field. (van Hardenbroek et al., 2013) went on to measure the δ13C of invertebrate remains from a sediment record (covering the past LOOKING FORWARD: KNOWLEDGE GAPS ∼1000 years) of a shallow thermokarst lake in northeast Siberia. Those taxa most sensitive to CH4 availability (Chironomini, To date, the majority of studies on CH4 in food webs have Tanytarsini, and Daphnia) exhibited the lowest δ13C values in solely concentrated on the stable carbon isotopes as a tracer. sediments deposited from ca AD 1250 to ca AD 1500, and However, equally evident to the very low and varying δ13C

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values in consumers part-fuelled by biogenic CH4 have been et al., 1987; Ings et al., 2012). More specifically, both Type I 15 low and highly variable δ N values; indeed, one of the most and II MOB can fix atmospheric N2 into ammonium and share striking patterns to emerge from studies involving chironomids similar pathways to oxidize ammonia/ammonium as autotrophic 13 and CH4 is the strong, positive relationship between δ C and ammonium oxidizing bacteria (Lee and Childress, 1994) and δ15N (Grey et al., 2004a) which appears to have some species- thus, are likely to exhibit correspondingly low δ15N values. specific basis (Kelly et al., 2004). These relationships appear However, ammonium oxidation rates are typically low and consistent and widespread (Figure 3) and while most likely high ammonium concentrations may inhibit CH4 oxidation. linked to assimilation of MOB, a test of the potential mechanisms In addition, some MOB can convert nitrate back to N2 and underpinning such low δ15N values in consumer tissues is such denitrifying methanotrophs may outcompete other MOB currently lacking. in nitrogen-rich, low oxygen environments (Stein and Klotz, In Grey et al. (2004a), it was postulated that nitrogen within 2011), which are characteristic of many of the lakes where low chironomid tubes may be continuously cycled between the δ13C and δ15N values in chironomids have been found (Jones larva and microbial consortia; for example, chironomids excrete et al., 2008). To examine the underlying causal mechanisms nitrogen in the form of ammonium directly into their tubes for the strong, consistent, and widespread relationship between and the overlying water (Devine and Vanni, 2002), and via chironomid δ13C and δ15N values, more research is required essential fractionation of ammonia, any microbial community to characterize the stable isotope values of potential nitrogen taking up that nitrogen source would be 15N-depleted (Macko sources, to measure potential N-fractionation by MOB, and

FIGURE 3 | Stable carbon and nitrogen isotope ratios of benthic chironomid larvae collected from stratifying lakes in Germany, England and Finland [data derived from Grey et al. (2004a), Deines et al. (2007a), Ravinet et al. (2010)]. Individuals were collected from a specific depth in each lake and on one date (except for Holzsee where the data are compiled from 12 sampling events in 1year). Species are Chironomus plumosus (filled black markers, solid line), Chironomus anthracinus (filled gray markers, dashed line), Propsilocerus jacuticus (Jyväsjärvi only; open triangle, dashed line), and Chironomus teniustylus (Halsjärvi only; open marker, dashed line). Lines are least squares regressions for illustrative purposes only.

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to use complementary methods such as molecular biomarker substantial oxidation of CH4 within the water column above profiling (PLFAs and 16S rRNA genes) of chironomid gut seeps off Svalbard, and carbon isotopic evidence that atmospheric contents. methane above those seeps is not influenced by contributions 13 While δ C values for dissolved CH4 are relatively easily from the seafloor source (Graves et al., 2015). Clearly then measuredinthelabaswellasinthefieldnowadays,andhenceare there must be MOB biomass accruing between the sediment available from a wide range of aquatic environments, more robust and the surface that could be incorporated into food webs, a end-member values for MOB are required if we are to improve pathway that is only likely to increase in importance if gas estimates for the quantitative contribution of CH4-carbon to total hydrate destabilization is promoted by warming of bottom carbon budgets and production figures for different ecosystems. waters. To date, such estimates have relied on some of the earliest simple Analyses of long-term data series from lakes demonstrate two-source mixing models (i.e., only using one stable isotope: that many are subject to increasing average water temperature carbon) by applying a range of trophic fractionation factors (Schindler et al., 1990; Hampton et al., 2008). While temperature for MOB (reported from a very small number of laboratory exerts a strong control on CH4 efflux via the physiological experiments) to values of CH4 gas to derive one end-member. stimulation of microbial metabolism (Gedney et al., 2004; Yvon- Direct measures of MOB δ13C from aquatic environments are Durocher et al., 2011), increasingly warm summer surface water badly needed. Currently, it is possible to measure the δ13C of temperatures may also increase the duration of stratification, MOB-specific PLFAs extracted from aquatic sediments, but how Schmidt stability and hypolimnetic oxygen depletion (e.g., these relate to the values from whole MOB cells still needs to Jankowski et al., 2006), all of which will have ramifications for be established. More laboratory studies of how carbon isotope CH4 dynamics and the routing of MDC into biomass (Jones and fractionation between CH4 and MOB may vary with different Grey, 2011). Some limited yet tantalizing empirical evidence for environmental and cell growth conditions would be extremely this arose from the physical manipulation of the depth of the useful, acknowledging that “controlling” every parameter even thermocline in a lake (compared to a nearby reference lake) by in the lab can be extremely difficult (e.g., Perga and Grey, installation of an impellor system (Forsius et al., 2010). As a 2010). consequence of deepening the thermocline, the dominant fish The geographic range of studies of MDC in food webs is still species, perch (Perca fluviatilis) were observed to become more rather limited. Within freshwaters, Jones et al. (2008) is the only 13C-depleted; a function of increased surface area of sediment paper to synthesize data from across a wide latitudinal gradient adjacent to oxygenated water ideal for chironomid uptake of and a distinct knowledge gap exists for the lower latitudes. MOB (route 3 in Figure 1), and the oxygenated water allowing Tropical regions are responsible for approximately half of the perch to forage on the benthos (Rask et al., 2010). Further estimated CH4 emissions from freshwater ecosystems to the manifestations of climate change, such as an increase in both the atmosphere, although they have been consistently under-sampled frequency and severity of storms, could affect both the strength (Bastviken et al., 2011). Indeed, the permanently stratified and duration of stratification in lakes, and increase the flux of (meromictic) Lake Kivu, within the western branch of the carbon from the catchment. Not only might erosion from the East African Rift, is one of the largest freshwater reservoirs of terrestrial ecosystem provide the substrate for methanogenesis dissolved methane (CH4) on Earth. Given the relatively high in aquatic ecosystems (e.g., Sanders et al., 2007), but increased magnitude of MOB production integrated over the entire water concentration and use of dissolved organic and inorganic carbon column reported by Morana et al. (2015) (equivalent to 16– in lakes and rivers (Schindler et al., 1997; Jones et al., 2001; 60% of the average photosynthetic primary production), and Worrall et al., 2004; Evans et al., 2005) will shift the balance the substantial contribution of MDC to the overall biomass toward heterotrophic rather than autotrophic functioning. Stable in the oxycline, suggest that MOB could potentially sustain a isotope tracers will remain key to unraveling the extent of MDC significant fraction of the pelagic food web in this lake. With use in such food webs in future research. few exceptions (like Lake Kivu), it should also be noted that the majority of studies have focused upon relatively small stratifying AUTHOR CONTRIBUTIONS stillwaters with strong oxygen gradients. The use of MDC in river food webs—substantial quantities of CH4 are oxidized in The author confirms being the sole contributor of this work and large riverine systems, including the Amazon and the Hudson approved it for publication. River (de Angelis and Scranton, 1993; Melack et al., 2004)— may prove to be a more widespread and significant ecosystem ACKNOWLEDGMENTS process than given credit at present (Trimmer et al., 2012). Whilst acknowledging that other chemosynthetic processes tend I have had many fruitful discussions on this topic over the past 15 to dominate in marine systems, the use of MDC at pelagic years or so, but particular thanks must be made to my long term boundaries, such as above the oxygen minimum zones of the collaborators Roger I. Jones and Mark Trimmer, as well as Peter various oceans, might well be locally important (but over vast Deines, Jari Syväranta, Oliver Heiri, Maarten van Hardenbroek, areas) to zooplankton as it is in stratifying lakes subject to Jos Schilder and Matt Wooller. This work was funded by the similar chemical gradients. There is very recent evidence for Natural Environment Research Council, UK (NE/H02235X/1).

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REFERENCES Deines, P., and Fink, P. (2011). The potential of methanotrophic bacteria to compensate for food quantity or food quality limitations in Daphnia. Aquat. Agasild, H., Zingel, P., Tuvikene, L., Tuvikene, A., Timm, H., Feldmann, T., et al. Microb. Ecol. 65, 197–206. doi: 10.3354/ame01542 (2014). Biogenic methane contributes to the food web of a large, shallow lake. Deines, P., and Grey, J. (2006). Site-specific methane production and subsequent Freshw. Biol. 59, 272–285. doi: 10.1111/fwb.12263 midge mediation within Esthwaite Water, UK. Archiv. Hydrobiol. 167, 317–334. Bade, D., Pace, M., Cole, J., and Carpenter, S. (2006). Can algal photosynthetic doi: 10.1127/0003-9136/2006/0167-0317 inorganic carbon isotope fractionation be predicted in lakes using existing Deines, P., Grey, J., Richnow, H. H., and Eller, G. (2007b). Linking larval models? Aquat. Sci. 68, 142–153. doi: 10.1007/s00027-006-0818-5 chironomids to methane: seasonal variation of the microbial methane Bastviken, D. (2009). “Methane,” in Encyclopedia of Inland Waters, ed G. E. Likens cycle and chironomid delta C-13. Aquat. Microb. Ecol. 46, 273–282. doi: (Oxford: Academic Press), 783–805. 10.3354/ame046273 Bastviken, D., Ejlertsson, J., Sundh, I., and Tranvik, L. (2003). Methane as a source Deines, P., Wooller, M. J., and Grey, J. (2009). Unravelling complexities in benthic of carbon and energy for lake pelagic food webs. Ecology 84, 969–981. doi: food webs using a dual stable isotope (hydrogen and carbon) approach. Freshw. 10.1890/0012-9658(2003)084[0969:MAASOC]2.0.CO;2 Biol. 54, 2243–2251. doi: 10.1111/j.1365-2427.2009.02259.x Bastviken, D., Tranvik, L. J., Downing, J. A., Crill, P. M., and Enrich-Prast, Devine, J. A., and Vanni, M. J. (2002). Spatial and seasonal variation in nutrient A. (2011). Freshwater methane emissions offset the continental carbon sink. excretion by benthic invertebrates in a eutrophic reservoir. Freshw. Biol. 47, Science 331, 50–50. doi: 10.1126/science.1196808 1107–1121. doi: 10.1046/j.1365-2427.2002.00843.x Battarbee, R. (1999). “The importance of palaeolimnology to lake restoration,” Devlin, S. P., Saarenheimo, J., Syvaranta, J., and Jones, R. I. (2015). Top in The Ecological Bases for Lake and Reservoir Management Developments consumer abundance influences lake methane efflux. Nat. Commun. 6:8787. in Hydrobiology, eds D. Harper, B. Brierley, A. D. Ferguson, and G. Phillips doi: 10.1038/ncomms9787 (Springer), 149–159. Dossena, M., Yvon-Durocher, G., Grey, J., Montoya, J. M., Perkins, D. M., Battin, T. J., Luyssaert, S., Kaplan, L. A., Aufdenkampe, A. K., Richter, A., and Trimmer, M., et al. (2012). Warming alters community size structure and Tranvik, L. J. (2009). The boundless carbon cycle. Nat. Geosci. 2, 598–600. doi: ecosystem functioning. Proc. R. Soc. B Biol. Sci. 279, 3011–3019. doi: 10.1038/ngeo618 10.1098/rspb.2012.0394 Belle, S., Parent, C., Frossard, V., Verneaux, V., Millet, L., Chronopoulou, P.-M., Doucett, R. R., Marks, J. C., Blinn, D. W., Caron, M., and Hungate, B. A. (2007). et al. (2014). Temporal changes in the contribution of methane-oxidizing Measuring terrestrial subsidies to aquatic food webs using stable isotopes of bacteria to the biomass of chironomid larvae determined using stable carbon hydrogen. Ecology 88, 1587–1592. doi: 10.1890/06-1184 isotopes and ancient DNA. J. Paleolimnol. 52, 215–228. doi: 10.1007/s10933- Duinen, G. A. V., Vermonden, K., Bodelier, P. L. E., Hendriks, A. J., Leuven, R. 014-9789-z S. E. W., Middelburg, J. J., et al. (2013). Methane as a carbon source for the Belle, S., Verneaux, V., Millet, L., Parent, C., and Magny, M. (2015). A case study food web in raised bog pools. Freshw. Sci. 32, 1260–1272. doi: 10.1899/12- of the past CH4 cycle in lakes by the combined use of dual isotopes (carbon and 121.1 hydrogen) and ancient DNA of methane-oxidizing bacteria: rearing experiment Eggermont, H., and Heiri, O. (2012). The chironomid-temperature relationship: and application to Lake Remoray (eastern France). Aquat. Ecol. 49, 279–291. expression in nature and palaeoenvironmental implications. Biol. Rev. 87, doi: 10.1007/s10452-015-9523-6 430–456. doi: 10.1111/j.1469-185X.2011.00206.x Bergstrom, I., Makela, S., Kankaala, P., and Kortelainen, P. (2007). Methane efflux Eller, G., Kanel, L. K., and Kruger, M. (2005). Cooccurrence of aerobic and from littoral vegetation stands of southern boreal lakes: an upscaled regional anaerobic methane oxidation in the water column of lake plusssee. Appl. estimate. Atmos. Environ. 41, 339–351. doi: 10.1016/j.atmosenv.2006.08.014 Environ. Microbiol. 71, 8925–8928. doi: 10.1128/AEM.71.12.8925-8928.2005 Bréas, O., Guillou, C., Reniero, F., and Wada, E. (2001). The global methane cycle: Estep, M. F., and Dabrowski, H. (1980). Tracing food webs with stable hydrogen isotopes and mixing ratios, sources and sinks. Isotopes Environ. Health Stud. 37, isotopes. Science 209, 1537–1538. doi: 10.1126/science.209.4464.1537 257–379. doi: 10.1080/10256010108033302 Evans, C. D., Monteith, D. T., and Cooper, D. M. (2005). Long-term Brooks, J. M., Kennicutt, M. C., Fisher, C. R., Macko, S. A., Cole, K., Childress, J. increases in surface water dissolved organic carbon: observations, possible J., et al. (1987). Deep-sea hydrocarbon seep communities - evidence for energy causes and environmental impacts. Environ. Pollut. 137, 55–71. doi: and nutritional carbon-sources. Science 238, 1138–1142. 10.1016/j.envpol.2004.12.031 Casper, P., Maberly, S. C., Hall, G. H., and Finlay, B. J. (2000). Fluxes of Finlay, J. C., Power, M. E., and Cabana, G. (1999). Effects of water velocity on methane and carbon dioxide from a small productive lake to the atmosphere. algal carbon isotope ratios: implications for river food web studies. Limnol. Biogeochemistry 49, 1–19. doi: 10.1023/A:1006269900174 Oceanogr. 44, 1198–1203. Colaço, A., Dehairs, F., and Desbruyères, D. (2002). Nutritional relations of deep- Forsius, M., Saloranta, T., Arvola, L., Salo, S., Verta, M., Ala-Opas, P., et al. (2010). sea hydrothermal fields at the Mid-Atlantic Ridge: a stable isotope approach. Physical and chemical consequences of artificially deepened thermocline in a Deep Sea Res. I Oceanogr. Res. Papers 49, 395–412. doi: 10.1016/S0967- small humic lake - a paired whole-lake climate change experiment. Hydrol. 0637(01)00060-7 Earth Syst. Sci. 14, 2629–2642. doi: 10.5194/hess-14-2629-2010 Cole, J. (2013). Freshwater in flux. Nat. Geosci. 6, 13–14. doi: 10.1038/ngeo1696 Frossard, V., Belle, S., Verneaux, V., Millet, L., and Magny, M. (2013). A Cole, J. J., Prairie, Y. T., Caraco, N. F., McDowell, W. H., Tranvik, L. J., Striegl, study of the δ13C offset between chironomid larvae and their exuvial head R. G., et al. (2007). Plumbing the global carbon cycle: integrating inland waters capsules: implications for palaeoecology. J. Paleolimnol. 50, 379–386. doi: into the terrestrial carbon budget. Ecosystems 10, 172–185. doi: 10.1007/s10021- 10.1007/s10933-013-9732-8 006-9013-8 Frossard, V., Verneaux, V., Millet, L., Magny, M., and Perga, M.-E. (2015). Changes Coleman, D. D., Risatti, J. B., and Schoell, M. (1981). Fractionation of carbon in carbon sources fueling benthic secondary production over depth and time: and hydrogen isotopes by methane-oxidizing bacteria. Geochim. Cosmochim. coupling Chironomidae stable carbon isotopes to larval abundance. Oecologia Acta 45, 1033–1037. 178, 603–614. doi: 10.1007/s00442-015-3225-6 Conrad, R. (2005). Quantification of methanogenic pathways using stable carbon Gedney, N., Cox, P. M., and Huntingford, C. (2004). Climate feedback isotopic signatures: a review and a proposal. Organ. Geochem. 36, 739–752. doi: from wetland methane emissions. Geophys. Res. Lett. 31:L20503. doi: 10.1016/j.orggeochem.2004.09.006 10.1029/2004GL020919 Cremona, F., Timm, H., Agasild, H., Tonno, I., Feldmann, T., Jones, R. I., et al. Gentzel, T., Hershey, A. E., Rublee, P. A., and Whalen, S. C. (2012). Net (2014). Benthic foodweb structure in a large shallow lake studied by stable sediment production of methane, distribution of methanogens and methane- isotope analysis. Freshw. Sci. 33, 885–894. doi: 10.1086/677540 oxidizing bacteria, and utilization of methane-derived carbon in an arctic lake. de Angelis, M. A., and Scranton, M. I. (1993). Fate of methane in the Hudson River Inland W. 2, 77–88. and Estuary. Global Biogeochem. Cycles 7, 509–523. doi: 10.1029/93GB01636 Graves, C. A., Steinle, L., Rehder, G., Niemann, H., Connelly, D. P., Lowry, D., Deines, P., Bodelier, P. L. E., and Eller, G. (2007a). Methane-derived carbon flows et al. (2015). Fluxes and fate of dissolved methane released at the seafloor at through methane-oxidizing bacteria to higher trophic levels in aquatic systems. the landward limit of the gas hydrate stability zone offshore western Svalbard. Environ. Microbiol. 9, 1126–1134. doi: 10.1111/j.1462-2920.2006.01235.x J. Geophys. Res. 120, 6185–6201. doi: 10.1002/2015JC011084

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Grey, J. (2006). The use of stable isotope analyses in freshwater ecology: Current FEMS Microbiol. Ecol. 28, 121–129. doi: 10.1111/j.1574-6941.1999. awareness. Pol. J. Ecol. 54, 563–584. tb00567.x Grey, J., and Deines, P. (2005). Differential assimilation of methanotrophic and Kankaala, P., Taipale, S., Grey, J., Sonninen, E., Arvola, L., and Jones, R. I. (2006). chemoautotrophic bacteria by lake chironomid larvae. Aquat. Microb. Ecol. 40, Experimental delta C-13 evidence for a contribution of methane to pelagic 61–66. doi: 10.3354/ame040061 food webs in lakes. Limnol. Oceanogr. 51, 2821–2827. doi: 10.4319/lo.2006.51. Grey, J., Jones, R. I., and Sleep, D. (2000). Stable isotope analysis of the origins of 6.2821 zooplankton carbon in lakes of differing trophic state. Oecologia 123, 232–240. Kankaala, P., Taipale, S., Li, L., and Jones, R. I. (2010). Diets of crustacean doi: 10.1007/s004420051010 zooplankton, inferred from stable carbon and nitrogen isotope analyses, in Grey, J., Kelly, A., and Jones, R. (2004a). High intraspecific variability in carbon lakes with varying allochthonous dissolved organic carbon content. Aquat. Ecol. and nitrogen stable isotope ratios of lake chironomid larvae. Limnol. Oceanogr. 44, 781–795. doi: 10.1007/s10452-010-9316-x 49, 239–244. doi: 10.4319/lo.2004.49.1.0239 Kankaala, P., Taipale, S., Nykaenen, H., and Jones, R. I. (2007). Oxidation, Grey, J., Kelly, A., Ward, S., Sommerwerk, N., and Jones, R. I. (2004b). Seasonal efflux, and isotopic fractionation of methane during autumnal turnover changes in the stable isotope values of lake-dwelling chironomid larvae in in a polyhumic, boreal lake. J. Geophys. Res. Biogeosci. 112:G02033. doi: relation to feeding and life cycle variability. Freshw. Biol. 49, 681–689. doi: 10.1029/2006jg000336 10.1111/j.1365-2427.2004.01217.x Kelly, A., Jones, R., and Grey, J. (2004). Stable isotope analysis provides Gudasz, C., Bastviken, D., Steger, K., Premke, K., Sobek, S., and Tranvik, fresh insights into dietary separation between Chironomus anthracinus L. J. (2010). Temperature-controlled organic carbon mineralization in lake and C-plumosus. J. N. Am. Benthol. Soc. 23, 287–296. doi: 10.1899/0887- sediments. Nature 466, 478–481. doi: 10.1038/nature09186 3593(2004)023<0287:SIAPFI>2.0.CO;2 Hampton, S. E., Izmest’Eva, L. R., Moore, M. V., Katz, S. L., Dennis, B., and Kohzu, A., Kato, C., Iwata, T., Kishi, D., Murakami, M., Nakano, S., et al. (2004). Silow, E. A. (2008). Sixty years of environmental change in the world’s largest Stream food web fueled by methane-derived carbon. Aquat. Microb. Ecol. 36, freshwater lake – Lake Baikal, Siberia. Glob. Change Biol. 14, 1947–1958. doi: 189–194. doi: 10.3354/ame036189 10.1111/j.1365-2486.2008.01616.x Lee, R. W., and Childress, J. J. (1994). Assimilation of inorganic nitrogen by marine Hanson, R. S., and Hanson, T. E. (1996). Methanotrophic bacteria. Microbiol. Rev. invertebrates and their chemoautotrophic and methanotrophic symbionts. 60, 439–471. Appl. Environ. Microbiol. 60, 1852–1858. Harrod, C., and Grey, J. (2006). Isotopic variation complicates analysis of trophic Lennon, J. T., Faiia, A. M., Feng, X., and Cottingham, K. L. (2006). Relative relations within the fish community of Plu beta see: a small, deep, stratifying importance of CO2 recycling and CH4 pathways in lake food webs along lake. Archiv. Hydrobiol. 167, 281–299. doi: 10.1127/0003-9136/2006/0167-0281 a dissolved organic carbon gradient. Limnol. Oceanogr. 51, 1602–1613. doi: He, R., Wooller, M. J., Pohlman, J. W., Catranis, C., Quensen, J., Tiedje, J. M., 10.4319/lo.2006.51.4.1602 et al. (2012). Identification of functionally active aerobic methanotrophs in Levin, L. A., and Michener, R. H. (2002). Isotopic evidence for chemosynthesis- sediments from an arctic lake using stable isotope probing. Environ. Microbiol. based nutrition of macrobenthos: the lightness of being at Pacific methane 14, 1403–1419. doi: 10.1111/j.1462-2920.2012.02725.x seeps. Limnol. Oceanogr. 47, 1336–1345. doi: 10.4319/lo.2002.47.5.1336 Heiri, O., Schilder, J., and van Hardenbroek, M. (2012). Stable isotopic analysis MacAvoy, S. E., Carney, R. S., Fisher, C. R., and Macko, S. A. (2002). Use of of fossil chironomids as an approach to environmental reconstruction: chemosynthetic biomass by large, mobile, benthic predators in the Gulf of state of development and future challenges. Fauna Norvegica 31, 7–18. doi: Mexico. Mar. Ecol. Prog. Ser. 225, 65–78. doi: 10.3354/meps225065 10.5324/fn.v31i0.1436 Macko, S. A., Fogel, M. L., Hare, P. E., and Hoering, T. C. (1987). Isotopic Hershey, A. E., Northington, R. M., Hart-Smith, J., Bostick, M., and Whalen, S. C. fractionation of nitrogen and carbon in the synthesis of amino acids by (2015). Methane efflux and oxidation, and use of methane-derived carbon by microorganisms. Chem. Geol. 65, 79–92. doi: 10.1016/0168-9622(87)90064-9 larval Chironomini, in arctic lake sediments. Limnol. Oceanogr. 60, 276–285. Mbaka, J. G., Somlai, C., Köpfer, D., Maeck, A., Lorke, A., and Schäfer, R. B. (2014). doi: 10.1002/lno.10023 Methane-derived carbon in the benthic food web in stream impoundments. Hessen, D. O., and Nygaard, K. (1992). Bacterial transfer of methane and detritus; PLoS ONE 9:e111392. doi: 10.1371/journal.pone.0111392 implications for the pelagic carbon budget and gaseous release. Arch. Hydrobiol. McCutchan J. H. Jr., Lewis W. M. Jr., Kendall, C., and McGrath, C. C. (2003). Beih. Ergebn. Limnol. 37, 139–148. Variation in trophic shift for stable isotope ratios of carbon, nitrogen, and Hobbie, J. E. (1988). A comparison of the ecology of planktonic sulfur. Oikos 102, 378–390. doi: 10.1034/j.1600-0706.2003.12098.x bacteria in fresh and salt water. Limnol. Oceanogr. 33, 750–764. doi: Melack, J. M., Hess, L. L., Gastil, M., Forsberg, B. R., Hamilton, S. K., Lima, 10.4319/lo.1988.33.4_part_2.0750 I. B. T., et al. (2004). Regionalization of methane emissions in the Amazon Ings, N. L., Hildrew, A. G., and Grey, J. (2012). ‘House and garden’: larval Basin with microwave remote sensing. Glob. Change Biol. 10, 530–544. doi: galleries enhance resource availability for a sedentary caddisfly. Freshw. Biol. 10.1111/j.1365-2486.2004.00763.x 57, 2526–2538. doi: 10.1111/fwb.12025 Morana, C., Borges, A. V., Roland, F. A. E., Darchambeau, F., Descy, J. P., Jankowski, T., Livingstone, D. M., Bührer, H., Forster, R., and Niederhauser, P. and Bouillon, S. (2015). Methanotrophy within the water column of a large (2006). Consequences of the 2003 European heat wave for lake temperature meromictic tropical lake (Lake Kivu, East Africa). Biogeosciences 12, 2077–2088. profiles, thermal stability, and hypolimnetic oxygen depletion: implications for doi: 10.5194/bg-12-2077-2015 a warmer world. Limnol. Oceanogr. 51, 815–819. doi: 10.4319/lo.2006.51.2.0815 Opsahl, S. P., and Chanton, J. P. (2006). Isotopic evidence for methane-based Jones, R., and Grey, J. (2004). Stable isotope analysis of chironomid larvae chemosynthesis in the upper floridan aquifer food web. Oecologia 150, 89–96. from some Finnish forest lakes indicates dietary contribution from biogenic doi: 10.1007/s00442-006-0492-2 methane. Boreal Environ. Res. 9, 17–23. Orphan, V. J., Hinrichs, K. U., Ussler, W., Paull, C. K., Taylor, L. T., Sylva, S. P., Jones, R. I., Carter, C. E., Kelly, A., Ward, S., Kelly, D. J., and Grey, J. et al. (2001). Comparative analysis of methane-oxidizing archaea and sulfate- (2008). Widespread contribution of methane-cycle bacteria to the diets of lake reducing bacteria in anoxic marine sediments. Appl. Environ. Microbiol. 67, profundal chironomid larvae. Ecology 89, 857–864. doi: 10.1890/06-2010.1 1922–1934. doi: 10.1128/AEM.67.4.1922-1934.2001 Jones, R. I., and Grey, J. (2011). Biogenic methane in freshwater food webs. Freshw. Parnell, A. C., Phillips, D. L., Bearhop, S., Semmens, B. X., Ward, E. J., Moore, Biol. 56, 213–229. doi: 10.1111/j.1365-2427.2010.02494.x J. W., et al. (2013). Bayesian stable isotope mixing models. Environmetrics 24, Jones, R. I., Grey, J., Quarmby, C., and Sleep, D. (2001). Sources and fluxes of 387–399. doi: 10.1002/env.2221 inorganic carbon in a deep, oligotrophic lake (Loch Ness, Scotland). Global Pel, R., Hoogveld, H., and Floris, V. (2003). Using the hidden isotopic Biogeochem. Cycles 15, 863–870. doi: 10.1029/2001GB001423 heterogeneity in phyto- and zooplankton to unmask disparity in trophic Jones, R. I., Grey, J., Sleep, D., and Arvola, L. (1999). Stable isotope analysis carbon transfer. Limnol. Oceanogr. 48, 2200–2207. doi: 10.4319/lo.2003.48. of zooplankton carbon nutrition in humic lakes. Oikos 86, 97–104. doi: 6.2200 10.2307/3546573 Perga, M.-E. (2010). Potential of delta C-13 and delta N-15 of cladoceran subfossil Kajan, R., and Frenzel, P. (1999). The effect of chironomid larvae on exoskeletons for paleo-ecological studies. J. Paleolimnol. 44, 387–395. doi: production, oxidation and fluxes of methane in a flooded rice soil. 10.1007/s10933-009-9340-9

Frontiers in Ecology and Evolution | www.frontiersin.org 12 February 2016 | Volume 4 | Article 8 Grey Identifying and Tracking Methane in Food Webs

Perga, M.-E. (2011). Taphonomic and early diagenetic effects on the C and N stable Stein, L. Y., and Klotz, M. G. (2011). Nitrifying and denitrifying pathways isotope composition of cladoceran remains: implications for paleoecological of methanotrophic bacteria. Biochem. Soc. Trans. 39, 1826. doi: studies. J. Paleolimnol. 46, 203–213. doi: 10.1007/s10933-011-9532-y 10.1042/BST20110712 Perga, M.-E., and Grey, J. (2010). Laboratory measures of isotope discrimination Stephen, E. M., Robert, S. C., Charles, R. F., and Stephen, A. M. (2002). Use of factors: comments on Caut, Angulo & Courchamp (2008,2009). J. Appl. Ecol. chemosynthetic biomass by large, mobile, benthic predators in the Gulf of 47, 942–947. doi: 10.1111/j.1365-2664.2009.01730.x Mexico. Mar. Ecol. Prog. Ser. 225, 65–78. doi: 10.3354/meps225065 Peterson, B. J., and Fry, B. (1987). Stable isotopes in ecosystem studies. Summons, R. E., Jahnke, L. L., and Roksandic, Z. (1994). Carbon isotopic Annu. Rev. Ecol. Syst. 18, 293–320. doi: 10.1146/annurev.es.18.110187. fractionation in lipids from methanotrophic bacteria: relevance for 001453 interpretation of the geochemical record of biomarkers. Geochim. Cosmochim. Prairie, Y., and del Giorgio, P. (2013). A new pathway of freshwater methane Acta 58, 2853–2863. doi: 10.1016/0016-7037(94)90119-8 emissions and the putative importance of microbubbles. Inland W. 3, 311–320. Taipale, S. J., Peltomaa, E., Hiltunen, M., Jones, R. I., Hahn, M. W., Biasi, C., et al. doi: 10.5268/IW-3.3.542 (2015). Inferring phytoplankton, terrestrial plant and bacteria bulk delta c-13 Rask, M., Verta, M., Korhonen, M., Salo, S., Forsius, M., Arvola, L., et al. (2010). values from compound specific analyses of lipids and fatty acids. PLoS ONE Does lake thermocline depth affect methyl mercury concentrations in fish? 10:e0133974. doi: 10.1371/journal.pone.0133974 Biogeochemistry 101, 311–322. doi: 10.1007/s10533-010-9487-5 Taipale, S., Kankaala, P., Hamalainen, H., and Jones, R. I. (2009). Seasonal shifts Rau, G. H., and Hedges, J. I. (1979). Carbon-13 depletion in a hydrothermal vent in the diet of lake zooplankton revealed by phospholipid fatty acid analysis. mussel: suggestion of a chemosynthetic food source. Science 203, 648–649. doi: Freshw. Biol. 54, 90–104. doi: 10.1111/j.1365-2427.2008.02094.x 10.1126/science.203.4381.648 Taipale, S., Kankaala, P., and Jones, R. I. (2007). Contributions of different organic Ravinet, M., Syvaranta, J., Jones, R. I., and Grey, J. (2010). A trophic pathway from carbon sources to Daphnia in the pelagic foodweb of a small polyhumic lake: biogenic methane supports fish biomass in a temperate lake ecosystem. Oikos results from mesocosm (DIC)-C-13-additions. Ecosystems 10, 757–772. doi: 119, 409–416. doi: 10.1111/j.1600-0706.2009.17859.x 10.1007/s10021-007-9056-5 Rinta, P., Bastviken, D., van Hardenbroek, M., Kankaala, P., Leuenberger, M., Taipale, S., Kankaala, P., Tiirola, M., and Jones, R. I. (2008). Whole-lake dissolved Schilder, J., et al. (2015). An inter-regional assessment of concentrations and inorganic C-13 additions reveal seasonal shifts in zooplankton diet. Ecology 89, δ13C values of methane and dissolved inorganic carbon in small European 463–474. doi: 10.1890/07-0702.1 lakes. Aquat. Sci. 77, 667–680. doi: 10.1007/s00027-015-0410-y Templeton, A. S., Chu, K.-H., Alvarez-Cohen, L., and Conrad, M. E. Roach, K. A., Tobler, M., and Winemiller, K. O. (2011). Hydrogen sulfide, bacteria, (2006). Variable carbon isotope fractionation expressed by aerobic and fish: a unique, subterranean food chain. Ecology 92, 2056–2062. doi: CH4-oxidizing bacteria. Geochim. Cosmochim. Acta 70, 1739–1752. doi: 10.1890/11-0276.1 10.1016/j.gca.2005.12.002 Sanders, I. A., Heppell, C. M., Cotton, J. A., Wharton, G., Hildrew, A. G., Trimmer, M., Grey, J., Heppell, C. M., Hildrew, A. G., Lansdown, K., Flowers, E. J., et al. (2007). Emission of methane from chalk streams has Stahl, H., et al. (2012). River bed carbon and nitrogen cycling: State potential implications for agricultural practices. Freshw. Biol. 52, 1176–1186. of play and some new directions. Sci. Total Environ. 434, 143–158. doi: doi: 10.1111/j.1365-2427.2007.01745.x 10.1016/j.scitotenv.2011.10.074 Sanseverino, A. M., Bastviken, D., Sundh, I., Pickova, J., and Enrich-Prast, A. Trimmer, M., Hildrew, A. G., Jackson, M. C., Pretty, J. L., and Grey, J. (2009). (2012). Methane carbon supports aquatic food webs to the fish level. PLoS ONE Evidence for the role of methane-derived carbon in a free-flowing, lowland river 7:e42723. doi: 10.1371/journal.pone.0042723 food web. Limnol. Oceanogr. 54, 1541–1547. doi: 10.4319/lo.2009.54.5.1541 Santer, B., Sommerwerk, N., and Grey, J. (2006). Food niches of cyclopoid Trimmer, M., Maanoja, S., Hildrew, A. G., Pretty, J. L., and Grey, J. (2010). copepods in eutrophic PluBsee determined by stable isotope analysis. Archiv. Potential carbon fixation via methane oxidation in well-oxygenated riverbed Hydrobiol. 167, 301–316. doi: 10.1127/0003-9136/2006/0167-0301 gravels. Limnol. Oceanogr. 55, 560–568. doi: 10.4319/lo.2009.55.2.0560 Sassen, R., Joye, S., Sweet, S. T., DeFreitas, D. A., Milkov, A. V., and MacDonald, Trimmer, M., Shelley, F. C., Purdy, K. J., Maanoja, S. T., Chronopoulou, P.-M., and I. R. (1999). Thermogenic gas hydrates and hydrocarbon gases in complex Grey, J. (2015). Riverbed methanotrophy sustained by high carbon conversion chemosynthetic communities, Gulf of Mexico continental slope. Organ. efficiency. ISME J. 9, 2304–2314. doi: 10.1038/ismej.2015.98 Geochem. 30, 485–497. doi: 10.1016/S0146-6380(99)00050-9 Van Dover, C. L., and Fry, B. (1994). Microorganisms as food resources Schilder, J., Bastviken, D., van Hardenbroek, M., Leuenberger, M., Rinta, P., at deep-sea hydrothermal vents. Limnol. Oceanogr. 39, 51–57. doi: Stoetter, T., et al. (2015a). The stable carbon isotopic composition of Daphnia 10.4319/lo.1994.39.1.0051 ephippia in small, temperate lakes reflects in-lake methane availability. Limnol. van Hardenbroek, M., Heiri, O., Grey, J., Bodelier, P. L. E., Verbruggen, F., Oceanogr. 60, 1064–1075. doi: 10.1002/lno.10079 and Lotter, A. F. (2010). Fossil chironomid delta(13) C as a proxy for past Schilder, J., Tellenbach, C., Möst, M., Spaak, P., van Hardenbroek, M., Wooller, methanogenic contribution to benthic food webs in lakes? J. Paleolimnol. 43, M. J., et al. (2015b). The stable isotopic composition of Daphnia ephippia 235–245. doi: 10.1007/s10933-009-9328-5 reflects changes in δ13C and δ18O values of food and water. Biogeosciences 12, van Hardenbroek, M., Heiri, O., Parmentier, F. J. W., Bastviken, D., Ilyashuk, B. P., 3819–3830. doi: 10.5194/bg-12-3819-2015 Wiklund, J. A., et al. (2013). Evidence for past variations in methane availability Schimmelmann, A. (2011). “Carbon, nitrogen and oxygen stable isotope ratios in in a Siberian thermokarst lake based on delta C-13 of chitinous invertebrate Chitin,” in Chitin Topics in Geobiology, ed N.S. Gupta (Springer), 81–103. remains. Quat. Sci. Rev. 66, 74–84. doi: 10.1016/j.quascirev.2012.04.009 Schindler, D. W., Beaty, K. G., Fee, E. J., Cruikshank, D. R., DeBruyn, E. R., Findlay, van Hardenbroek, M., Heiri, O., Wilhelm, M. F., and Lotter, A. F. (2011). How D. L., et al. (1990). Effects of climatic warming on lakes of the central boreal representative are subfossil assemblages of Chironomidae and common benthic forest. Science 250, 967–970. doi: 10.1126/science.250.4983.967 invertebrates for the living fauna of Lake De Waay, the Netherlands? Aquat. Sci. Schindler, D. W., Curtis, P. J., Bayley, S. E., Parker, B. R., Beaty, K. G., and Stainton, 73, 247–259. doi: 10.1007/s00027-010-0173-4 M. P. (1997). Climate-induced changes in the dissolved organic carbon budgets van Hardenbroek, M., Leuenberger, M., Hartikainen, H., Okamura, B., and of boreal lakes. Biogeochemistry 36, 9–28. doi: 10.1023/A:1005792014547 Heiri, O. (2016). Bryozoan stable carbon and hydrogen isotopes: relationships Schmitz, O., Raymond, P., Estes, J., Kurz, W., Holtgrieve, G., Ritchie, M., between the isotopic composition of zooids, statoblasts and lake water. et al. (2014). Animating the carbon cycle. Ecosystems 17, 344–359. doi: Hydrobiologia 765, 209–223. doi: 10.1007/s10750-015-2414-y 10.1007/s10021-013-9715-7 van Hardenbroek, M., Lotter, A. F., Bastviken, D., Duc, N. T., and Heiri, O. Shelley, F., Abdullahi, F., Grey, J., and Trimmer, M. (2015). Microbial methane (2012). Relationship between d13C of chironomid remains and methane flux in cycling in the bed of a chalk river: oxidation has the potential to match Swedish lakes. Freshw. Biol. 57, 166–177. doi: 10.1111/j.1365-2427.2011.02710.x methanogenesis enhanced by warming. Freshw. Biol. 60, 150–160. doi: Vuorio, K., Meili, M., and Sarvala, J. (2006). Taxon-specific variation in the stable 10.1111/fwb.12480 isotopic signatures (δ13C and δ15N) of lake phytoplankton. Freshw. Biol. 51, Shelley, F., Grey, J., and Trimmer, M. (2014). Widespread methanotrophic primary 807–822. doi: 10.1111/j.1365-2427.2006.01529.x production in lowland chalk rivers. Proc. R. Soc. B Biol. Sci. 281:20132854. doi: Werne, J. P., Baas, M., and Sinninghe Damsté, J. S. (2002). Molecular isotopic 10.1098/rspb.2013.2854 tracing of carbon flow and trophic relationships in a methane-supported

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benthic microbial community. Limnol. Oceanogr. 47, 1694–1701. doi: Yvon-Durocher, G., Jones, J. I., Trimmer, M., Woodward, G., and Montoya, 10.4319/lo.2002.47.6.1694 J. M. (2010). Warming alters the metabolic balance of ecosystems. Whiticar, M. J. (1999). Carbon and hydrogen isotope systematics of bacterial Philos. Trans. R. Soc. B Biol. Sci. 365, 2117–2126. doi: 10.1098/rstb. formation and oxidation of methane. Chem. Geol. 161, 291–314. doi: 2010.0038 10.1016/S0009-2541(99)00092-3 Yvon-Durocher, G., Montoya, J. M., Woodward, G., Jones, J. I., Whiticar, M. J., Faber, E., and Schoell, M. (1986). Biogenic methane and Trimmer, M. (2011). Warming increases the proportion of formation in marine and freshwater environments: CO2 reduction vs. acetate primary production emitted as methane from freshwater mesocosms. fermentation—Isotope evidence. Geochim. Cosmochim. Acta 50, 693–709. doi: Glob. Change Biol. 17, 1225–1234. doi: 10.1111/j.1365-2486.2010. 10.1016/0016-7037(86)90346-7 02289.x Wooller, M., Pohlman, J., Gaglioti, B., Langdon, P., Jones, M., Walter Anthony, K., et al. (2012). Reconstruction of past methane availability in an Arctic Alaska Conflict of Interest Statement: The author declares that the research was wetland indicates climate influenced methane release during the past ∼12,000 conducted in the absence of any commercial or financial relationships that could years. J. Paleolimnol. 48, 27–42. doi: 10.1007/s10933-012-9591-8 be construed as a potential conflict of interest. Worrall, F., Harriman, R., Evans, C. D., Watts, C. D., Adamson, J., Neal, C., et al. (2004). Trends in dissolved organic carbon in UK rivers and lakes. Copyright © 2016 Grey. This is an open-access article distributed under the terms Biogeochemistry 70, 369–402. doi: 10.1007/s10533-004-8131-7 of the Creative Commons Attribution License (CC BY). The use, distribution or Yasuno, N., Shikano, S., Muraoka, A., Shimada, T., Ito, T., and Kikuchi, E. (2012). reproduction in other forums is permitted, provided the original author(s) or licensor Seasonal increase of methane in sediment decreases δ13C of larval chironomids are credited and that the original publication in this journal is cited, in accordance in a eutrophic shallow lake. Limnology 13, 107–116. doi: 10.1007/s10201-011- with accepted academic practice. No use, distribution or reproduction is permitted 0360-6 which does not comply with these terms.

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